The extracellular matrix (ECM) as it occurs in vivo plays a crucial role in the organization, homeostasis, and function of tissues and organs. Continuous communication between cells and their surrounding ECM environment orchestrates critical processes such as the acquisition and maintenance of differentiated phenotypes during embryogenesis, the development of form (morphogenesis), angiogenesis, wound healing, and even tumor metastasis. Both biochemical and biophysical signals from the ECM modulate fundamental cellular activities including adhesion, migration, proliferation, differential gene expression, and programmed cell death. The realization of the significance of the ECM to the organization, homeostasis, and function of tissues and organs has led to a renewed interest in characterizing ECM constituents and the relationship of these constituents to the functioning of the ECM.
ECMs, including various basement membrane tissues and other extracellular matrix tissues obtained from natural sources and matrices formed from isolated ECM components, can be utilized as tissue graft compositions for remodeling tissues in vivo or for in vitro applications. Complex scaffolds representing combinations of isolated extracellular matrix components in a natural or processed form are commercially available and can be used as tissue graft compositions (e.g., Human Extracellular Matrix (Becton Dickinson) and MATRIGEL®). Basement membrane tissues and other extracellular matrix tissues, such as tissue material derived from submucosal tissues, harvested from warm-blooded vertebrates have also shown great promise as unique graft materials for inducing the repair of damaged or diseased tissues in vivo, and for inducing the proliferation of cells in vitro.
In accordance with the invention, purified collagen can be used to produce an engineered ECM material prepared under conditions that regulate the polymerization of collagen in a controlled manner. This result is more difficult to achieve with existing intact or processed ECMs from natural sources and with ECMs and collagen preparations from commercial sources.
In the literature, there are known methods for isolating collagen from a variety of tissues, e.g., placenta, bladder, animal tails, and skin, and using the isolated material to reconstitute collagenous matrices. These collagenous matrices may have applications as graft materials for inducing the repair of damaged or diseased tissues in vivo, and for inducing the proliferation and other fundamental behavior of cells in vitro. The molecular forces that orchestrate the self assembly of soluble, monomeric collagen into higher ordered structures are weak so their assembly can easily turn into an unstructured aggregation of misfolded proteins. As reported herein, modifying the conditions used to isolate collagen results in collagen preparations with properties that enhance the rate at which the collagen polymerizes and that enhance the microstructural and mechanical properties of the collagen upon polymerization (e.g., the mechanical integrity of the engineered ECM that is formed upon collagen polymerization). The methods of collagen isolation and the collagen compositions described herein allow for the controlled alteration of the microstructural and subsequent mechanical properties of a resulting engineered ECM for such uses as graft compositions for inducing the repair of damaged or diseased tissues in vivo, and for inducing the proliferation and other fundamental behaviors of cells in vitro.
In one embodiment, a method for isolating collagen is provided. The method comprises the steps of obtaining a collagen-containing source material, comminuting the source material, mixing the comminuted source material with an extraction solution, extracting the comminuted source material to form a soluble fraction and an insoluble fraction, obtaining the insoluble fraction, extracting the collagen from the insoluble fraction to form a soluble collagen fraction, precipitating the collagen from the soluble collagen fraction, and resuspending the precipitate in an aqueous solution wherein the aqueous solution used to resuspend the collagen precipitate is an acidic solution. An isolated collagen composition prepared by this method is also provided.
In various embodiments of the embodiment described in the preceding paragraph: 1) the collagen-containing source material is selected from the group consisting of placental tissue, bladder tissue, intestinal tissue, alimentary tract tissue, ovarian tissue, pericardial tissue, animal tail tissue, liver tissue, skin tissue, and any other suitable collagen-containing source material, 2) the collagen-containing source material is selected from the group consisting of bladder tissue and skin tissue, 3) the collagen-containing source material is porcine skin tissue, 4) the source material is frozen in liquid nitrogen prior to the comminuting step, 5) the source material is frozen in liquid nitrogen during the comminuting step, 6) the source material is frozen in liquid nitrogen prior to and during the comminuting step, 7) the source material is frozen at a temperature of −20° C. or below prior to or during the comminuting step, 8) the source material is frozen at a temperature of −40° C. or below prior to or during the comminuting step, 9) the source material is frozen at a temperature of −60° C. or below prior to or during the comminuting step, 10) the source material is frozen at a temperature of −80° C. or below prior to or during the comminuting step, 11) the mixing step is performed by blending or stirring, 12) the mixing step is performed by stirring, 13) the soluble collagen fraction is not filtered between the step of extracting the collagen from the insoluble fraction to form the soluble collagen fraction and the step of precipitating the collagen from the soluble collagen fraction, 14) the method further comprises the step of lyophilizing the collagen precipitate, 15) the method further comprises the step of polymerizing the collagen prior to or after lyophilization, 16) the collagen is precipitated by dialysis against a buffered solution, and 17) the dialysis tubing has a molecular weight cut-off of about 12,000 to about 14,000. In an alternative embodiment, any of these steps can be used in any combination.
In one embodiment, a method for engineering matrices with enhanced polymerization characteristics is provided. The method comprises the steps of obtaining collagen oligomers, polymerizing the collagen oligomers, and forming the engineered matrices with enhanced polymerization characteristics. An engineered matrix prepared by this method is also provided.
In other embodiments, the enhanced polymerization characteristics are selected from the group consisting of an enhanced rate of polymerization, a reduced lag time for polymerization, and an enhanced mechanical integrity, and the enhanced mechanical integrity is selected from the group consisting of strength and stiffness. In yet another embodiment, the collagen oligomers polymerize with a maximal t ½ selected from the group consisting of about 3 minutes, about 2.5 minutes, about 2.0 minutes, about 1.5 minutes, and about 1 minute.
In other illustrative embodiments, the collagen oligomers are obtained by isolation from a collagen-containing source material that is a tissue naturally enriched with collagen oligomers, the collagen oligomers are obtained by isolating and then chemically cross-linking collagen, or the collagen oligomers are isolated from a diseased tissue or a genetically-modified tissue.
In still another embodiment, an engineered matrix is provided. The matrix comprises a predetermined percentage of collagen oligomers based on total isolated collagen used to make the engineered matrix. In various embodiments, the collagen is isolated from a tissue naturally enriched with collagen oligomers, the collagen oligomers are isolated from a diseased tissue or a genetically-modified tissue, or the collagen oligomers are obtained by isolating and then chemically cross-linking collagen.
In another illustrative embodiment, the predetermined percentage of collagen oligomers is selected from the group consisting of about 0.5% to about 100%, about 1.0% to about 100%, about 2% to about 100%, about 3% to about 100%, about 5% to about 100%, about 10% to about 100%, about 15% to about 100%, about 20% to about 100%, about 30% to about 100%, about 40% to about 100%, about 50% to about 100%, about 60% to about 100%, about 70% to about 100%, about 80% to about 100%, about 90% to about 100%, about 95% to about 100%, and about 100%. In still another embodiment, the engineered matrix further comprises a predetermined percentage of isolated collagen monomers.
In another aspect, a graft composition is provided. The graft composition comprises an engineered matrix comprising collagen oligomers wherein the matrix has a predetermined percentage of collagen oligomers based on total isolated collagen used to make the engineered matrix. In one embodiment, the predetermined percentage of collagen oligomers is selected from the group consisting of about 0.5% to about 100%, about 1.0% to about 100%, about 2% to about 100%, about 3% to about 100%, about 5% to about 100%, about 10% to about 100%, about 15% to about 100%, about 20% to about 100%, about 30% to about 100%, about 40% to about 100%, about 50% to about 100%, about 60% to about 100%, about 70% to about 100%, about 80% to about 100%, about 90% to about 100%, about 95% to about 100%, and about 100%. In various embodiments, the collagen oligomers are isolated from a diseased tissue or a genetically-modified tissue, the collagen oligomers are obtained by isolating and then chemically cross-linking collagen, or the collagen oligomers are isolated from a natural tissue enriched with collagen oligomers. In yet another embodiment, the graft composition further comprises a predetermined percentage of collagen monomers based on total isolated collagen used to make the engineered matrix.
Surprisingly, the inclusion of increased amounts of collagen oligomers in a collagen composition may increase the rate of polymerization, facilitate hierarchical assembly of component collagen fibrils, and enhance mechanical properties (e.g., strength and stiffness).